DESCRIPTION (taken from the application) Steroid hormones are ubiquitous regulators of physiologic process that are mandatory for the survival of the individual and the propagation of the species. The first, rate limiting and hormonally regulated step in the biosynthesis of all steroid hormones is the conversion of cholesterol to pregnenolone by the mitochondrial cytochrome P450scc system. P450scc activity is regulated chronically by the tissue-specific and hormonally regulated transcription of the P450scc gene. However, the acute, rapid, regulation of steroidogenesis, which occurs in 10-15 minutes as in the stress responses, is regulated at the level of cholesterol entry into mitochondria. This cholesterol flow is regulated by the Steroidogenic Acute Regulatory protein (StAR). StAR is found in all steroidogenic tissues that exhibit an acute response, and StAR mutations cause potentially lethal congenital lipoid adrenal hyperplasia. However, some tissues that make steroids (placenta, brain) lack StAR, and the mechanism of StAR's action is unknown. This StAR-independent steroidogenesis may be associated with MLN64, a ubiquitously expressed protein that is structurally related to StAR. The present project will expand our knowledge of the structure and function of StAR and MLN64 through five aims 1) Establish an in vitro assay for the cholesterol-transport activity of StAR and MLN-64, 2) Adapt our established procedure for the bacterial expression and purification of N-62 homologue of StAR to the StAR-like domain of MLN-64, N234 MLN-64 3) Characterize the folding of N-234 MLN-64 by circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), and fluorescence spectroscopy, 4) Compare the dynamics and folding of N-62 StAR and N-234 MLN-64 by deuterium exchange mass spectrometry, and 5) Determine the structure of N234 MLN-64 by multidimensional high resolution nuclear magnetic resonance (NMR) spectroscopy. Fulfilling these aims will provide the structural information needed to develop a detailed understanding of how StAR and MLN64 regulate cholesterol movement from the outer to inner mitochondrial membrane.